The traditional approach for improving microbial production of natural products, such as amino acids, antibiotics, etc., includes altering key structural or regulatory genes of the biosynthetic pathway followed by measuring the amount of desired product that is produced. Each change then reveals the presence or absence of a bottleneck and, based on those results, the next gene is deleted or overexpressed, and the cycle repeats until product titers/yields can no longer be improved substantially. Although this step-wise approach can yield improvements in flux through these pathways, it is a tedious and time-consuming strategy, given that metabolic pathways tend to be well balanced and rarely does a single change increase flux dramatically. Indeed, some bottlenecks will not be revealed until others are relieved. This process typically leads to the identification of local yield maxima, but not the global optimal yield.
These challenges are particularly evident in efforts to engineer Escherichia coli to produce high yields of aromatic amino acids. With advances in metabolic engineering and discovery of novel biosynthetic pathways in plants, aromatic amino acids, which have been important commodities used as animal feeds, food additives, and supplements, can also serve as precursors to a variety of commercially valuable molecules and pharmaceutical drugs (Gosset, G. Curr Opin Biotechnol 20:651-658, 2009; and Sprenger, G A. Appl Microbiol Biotechnol 75:739-749, 2007). It has recently been shown that L-tyrosine over-producing strains of Escherichia coli grown on glucose can be used to produce biopolymer starting materials such as p-hydroxycinnamic acid and p-hydroxystyrene, and drug precursors such as reticuline, an important intermediate in biosyntheses of benzylisoquinoline alkaloids (Minami, H J et al. Proc Natl Acad Sci USA 105:7393-7398, 2008; Sariaslani, F S et al. Annu Rev Microbiol 61:51-69, 2007; and Sato, F T et al. Curr Pharm Biotechnol 8:211-218, 2007). Yet, of the three aromatic amino acids derived from the shikimate pathway, the L-tyrosine yield has been shown to be the lowest. While L-tyrosine titers of over 50 g/L can be produced using E. coli in a 200-L bioreactor by improving the fermentation and isolation steps (Patnaik, R et al. Biotechnol Bioeng 99:741-752, 2008), the production strain only yielded about 0.10 gram of L-tyrosine per gram of glucose (Olson, M M et al. Microbiol Biotechnol 74:1031-1040, 2007). Accordingly, a need exists for further improvements in L-tyrosine yields to make the process as economically competitive as the processes used to synthesize other amino acids, such as L-lysine, L-glutamate, and L-alanine (Ikeda, M. Appl Microbiol Biotechnol 69:615-626, 2006; and Leuchtenberger, W et al. Appl Microbiol Biotechnol 69:1-8, 2005).
Another problem with increasing L-tyrosine yields is that despite a vast wealth of literature accumulated over the past thirty years pertaining to the enzymatic activities and expression properties of the shikimate pathway, the pathway remains difficult to engineer (Bongaerts, J et al. Metab Eng 3:289-300, 2001; Gosset, G. Curr Opin Biotechnol 20:651-658, 2009; Herrmann, K M and Weaver, L M. Physiol Plant Mol Biol 50:473-503, 1999; Ikeda, M. Appl Microbiol Biotechnol 69:615-626, 2006; and Sprenger, G A. Appl Microbiol Biotechnol 75:739-749, 2007). Previous L-tyrosine engineering work has most often focused on the transcriptional deregulation of the tyrR and/or trpR regulons, followed by removing the feedback inhibition on two key enzymes, 3-deoxy-D-arabino-heptulosonate (DAHP) synthase (AroG), which catalyzes the first committed step to the shikimate pathway, and the dual function chorismate mutase/prephenate dehydrogenase (TyrA), which catalyzes the first two steps in L-tyrosine biosynthesis from chorismate (Lutke-Eversloh, T and Stephanopoulos, G. Appl Microbiol Biotechnol 75:103-110, 2007; and Olson, M M et al. Appl Microbiol Biotechnol 74:1031-1040, 2007). Co-expression of the rate-limiting enzymes shikimate kinase (AroK or AroL) and quinate/shikimate dehydrogenase (YdiB), and deletion of the L-phenylalanine branch of the aromatic amino acid biosynthetic pathway have been shown to increase the L-tyrosine production (Gavini, N and Pulakat, L. J Bacteriol 173:4904-4907, 1991; Lutke-Eversloh, T and Stephanopoulos, G. Appl Microbiol Biotechnol 75:103-110, 2007; and Olson, M M et al. Appl Microbiol Biotechnol 74:1031-1040, 2007). Furthermore, overexpression of phosphoenolpyruvate synthase (PpsA) and transketolase A (TktA), altering glucose transport and use of other carbon sources, such as xylose and arabinose, have also been shown to increase the precursor pools to the shikimate pathway (Ahn, J O et al. J Microbiol Biotechnol 18:1773-1784, 2008; Daraths, K et al. Journal of the American Chemical Society 114:3956-3962, 1992; Li, K and Frost, J W. Biotechnol Prog 15:876-883, 1999; Lutke-Eversloh, T and Stephanopoulos, G. Appl Microbiol Biotechnol 75:103-110, 2007; Patnaik, R and Liao, J C. Appl Environ Microbiol 60:3903-3908, 1994; Yi, L et al. Biotechnol Prog 19:1450-1459, 2003; and Yi, J et al. Biotechnol Prog 18:1141-1148, 2002. In these previous studies, gene expression was modified for only a few candidates of the L-tyrosine pathway at a time, and a large number of strains had to be screened to circumvent bottlenecks. However, a problem with these approaches is that while one pathway bottleneck may be eliminated, a new bottleneck may be introduced somewhere else along the pathway.
Accordingly, there exists a need for improved approaches for engineering new microbial strains that contain all the genes necessary for producing amino acids and stable amino acid intermediates that reduces biosynthetic pathway bottlenecks and optimized the production of the amino acids and stable amino acids intermediates, thus improving overall product yield.